CAROTENE DEGRADATION
Reaction of Carotene with Nitrite Solutions
DONALD 1. PUGH and G. B. GARNER Department of Agricultural Chemistry, University of Missouri, Columbia, Mo.
Nitrite solutions of low pH, which would include any chemical changes in nitrite resulting from acidic conditions, are shown to cause destruction of carotene. Vitamin A alcohol is also readily destroyed, while p-apo-8’- and p-apo-l2’-carotenal are more resistant to attack by these solutions. Since carotene is a major source of vitamin A for cattle, it may be destroyed b y nitrite before absorption and conversion can take place. The recent apparent increase in vitamin A requirement for cattle fed forages high in nitrate may conceivably b e explained on this basis.
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1945, it’ilson ( 9 ) demonstrated the disappearance of the yellow color from carotene solutions when they \+.ere shaken with nitrous acid. H e had earlier reviewed the literature relating to the occurence of nitrite or nitrous acid in nature ( 8 ) . H e also determined the nitrate content of the expressed sap of a number of genera and species of plants, finding amounts ranging from a trace to 10,000 p.p.m. (70). Flynn et al. (3) found high levels of nitrate in corn plants, particularly after nitrogen fertilization and drouth conditions. Using artificial rumens, Barnett and Bowman (7) have shown that nitrate can be reduced to nitrite by rumen liquor. Lewis (6) found nitrite accumulation in vivo under certain conditions of nitrate feeding. Case (2) has discussed the deleterious effects of both of these substances on animals, and investigation by O’Dell et al. (7) showed that dietary nitrite enhanced vitamin A deficiency in rats and reduced vitamin A storage when carotene was fed. Garner (4) reported a n increase in the vitamin A requirement of cattle when nitrate-containing rations were being fed. The following in vitro experiments were run to investigate further the effect of both nitrite and hydrogen ion concentration on the destruction of carotene, since nitrate has been found nondestructive a t p H 1 to 7 by this laboratory. N
Procedure
A Latin square experimental design was used in these experiments. The variables involved were the molar ratio of nitrite to carotenoid pigment and the pH of the solution. In the first series of experiments, nitrite to carotene ratios were varied from 1 : 1 to 100: 1 and were used at each p H from 1 through 7. In the
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second series of experiments, @-carotene, P-apo- 8 ’-carotenal, @-apo-12’-carotenal: and vitamin 4 alcohol were subjected to similar conditions. Test tubes measuring 13 X 100 mm. were used in these experiments. The total volume per tube was 7 m1.-2 ml. of stock carotenoid solution. 2 ml. of the appropriate sulfuric acid solution, and 3 ml. of an appropriate dilution of the stock K N 0 2solution. The reagents were mixed at room temperature, then incubated 4 hours at 37’ C. The absorbance was determined by the use of a Beckman Model DU spectrophotometer. The blank was composed of the original reagent mixture with double-distilled water replacing the KN02 portion of the solution. Bcarotene, @-apo-8’-carotenal?and 8-apo12’-carotenal were read a t 450 mp while vitamin A alcohol was read a t 325 mp. Stock Solutions. Potassium nitrite stock solution was prepared to contain 126.48 pg. of K S 0 2 per ml. After drying overnight, 7.925 grams of KN02 were weighed into a 500-ml. volumetric flask. and made to volume with doubledistilled water. An aliquot of 7.98 ml. of this solution was diluted to 1 ltier to give the final desired concentration (126.48 pg. per ml. or 148.8 X IO-* moles per ml.). The carotenoid (or vitamin A alcohol) stock solutions were prepared as an aqueous dispersion with Iy0 Tween 80 (Atlas Powder Co., Wilmington, Del.). Crystalline p-carotene (Nutritional Biochemicals Corp.), @-apo-8’-carotenal,0apo-l2’-carotenal (Hoffmann-La Roche, Inc.), and vitamin A alcohol (Distillation Products Industries) were used. Each solution was prepared to contain a final concentration of 3.72 X moles of dissolved reactant per ml. The crystalline product (4 mg. of /3-carotene, 3.1
AGRICULTURAL A N D FOOD CHEMISTRY
mg. of @-apo-8’-carotenal, 3.2 mg. of @-apo-12’-carotenal, 2.1 mg. of vitamin A alcohol) was dissolved in approximately 40 ml. of absolute ethanol by warming. The ethanolic solution was added slowly, with mixing, to 100 ml. of warm 1% Tween 80 solution. -4dditional 1% Tween 80 solution was used to bring the solution to volume in a 200 ml. volumetric flask. A series of sulfuric acid solutions was prepared, ranging in p H from 0.5 to 5.5. Fresh, double-distilled water was used for the solution of p H 7. T h e p H of the sulfuric acid solutions was checked with a p H meter and adjusted when necessary. When diluted to the final concentration in the reaction. these solutions provided a calculated p H of 1 through 7.
Results and Discussion Figure 1 shows the results of triplicate experiments on the destruction of p-carotene by nitrite. The p H of the solutions was not a factor in the destruction of carotene in the control series, while destruction by nitrite depended markedly on the p H of the system. The amount of destruction at pH 5 to 7 at any given nitrite to carotene ratio was much less than at lower p H values. The destruction of carotene was greatest at p H 1 to 3 with a tendency toxvard a plateau at p H 2 to 3. At high nitrite to carotene ratios, the amount of destruction between p H 2 and 3 tended to equal that a t p H 1. An intermediate amount of destruction occurred a t p H 4. Previous work (5) on degradation of carotenoids would indicate that one possible pathway of degradation is through the apo-carotenals. Figure 2 shows the results of duplicate experiments comparing the destruction of @-carotene, P-apo-8 ’-carotenal, @-apo-
0.8
0.7
0.6
0.:
B
0..
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0.
0
04 6
7
5
4
3
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8-
PH
Figure 1 . Destruction of @-carotene(4 hours a t function of pH and nitrite concentration
37" C.) as a
Molar nitrite to carotene ratios: A-control, B - l : l , C-4:1, D-6:1, E-8:1, 6 1 0 : 1 , G-12: 1 , H-16: 1 , 1-20: 1 , J-40: 1 , and K-1 00: 1
12'-carotenal, and vitamin A alcohol. This figure compares results a t pH 2 only. The two carotenals are more stable in the presence of nitrite than is 8-carotene, 6-apo-12 '-carotenal being more stable than P-apo-8 '-carotenal. Yitamin A alcohol was more susceptible to degradation by nitritt- than the other three substances. In silage, a pH of 4 is normal. .4ny nitrite formed a t this pH would readily destroy carotene because of a n extended reaction period and elevated temperatures. I n the true stomach of animals (pH 2 to 3) the presence of nitrate along with dietary carotene could lead to a low vitamin A status. These data. although ~
Figure 2, Comparison of the destruction of three provitamin A compounds and vitamin A alcohol by nitrite a t pH 2 Molar nitrite to carotenoid ratios: C - 4 : 1 , D-10:1, and E-20: 1
collected using pure compounds and relatively simple reaction mixtures, suggest a possible reason for the apparent increase in vitamin A requirement of cattle fed forages containing a high level of nitrate.
literature Cited
(1) Barnett, A. J. G., Bowman. I. B. R.. J . Sci. Food Agr. 8, 243 (1957). (2) Case, A . D., J . Am. Vet. Med. Assoc. 130, 323 (1957). (3) Flynn. L. M . , Gehrke, C. W., Muhrer, M. E., Smith, G. E., Zuber, M. S.. Missouri Cniu. Agr. Expt. Sta. Res. Bull. 620 (1957).
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solid bar-control, A-0.5: 1 , B-1 : 1 ,
(4) Garner. G. B., Zbzd.. 708 (1958). (5) Karrer, P., Jucker, E., "Carotenoids," trans. Ernest A. Braude, p. 144, Elsevier, Kew York, 1950. (6) Lewis, D., Biochern. J . 48, 175 (1951). (7) O'Dell, B. L., Erek, Z., Flynn, L., Garner, G. B., Muhrer, M. E., J . Animal Sci. 19, 1280 (1960). (8) LVilson. J. K., Cornell Univ. Agr. Expt. Sta. Mem. 253, 1 (1943). (9) Zbid., 271, 1 (1945). (10) Wilson, J. K., J . A m . Soc. Agron. 35, 279 (1943). Received for reuiew August 27, 7962. -4ccepted December 18, 1962. Contribution from the LWMissouri Alricultural Experiment Station Journal Series .\'a. 2493. Approved by the director.
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1963
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